Gastroenterology

Gastroenterology

Volume 145, Issue 6, December 2013, Pages 1300-1311
Gastroenterology

Original Research
Full Report: Basic and Translational—Alimentary Tract
Bile Acids Increase Levels of MicroRNAs 221 and 222, Leading to Degradation of CDX2 During Esophageal Carcinogenesis

https://doi.org/10.1053/j.gastro.2013.08.008Get rights and content

Background & Aims

Bile reflux contributes to development of Barrett's esophagus (BE) and could be involved in its progression to esophageal adenocarcinoma (EAC). We investigated whether bile acids affect levels or functions of microRNAs (MIRs) 221 and 222, which bind to the 3′-UTR of p27Kip1 messenger RNA to inhibit its translation. Reduced p27Kip1 increases degradation of the transcription factor CDX2; levels of CDX2 have been reported to decrease during progression of BE to EAC.

Methods

We used quantitative reverse transcriptase polymerase chain reaction to compare levels of MIRs 221 and 222 and immunohistochemistry to compare levels of p27Kip1 and CDX2 proteins in areas of BE and EAC from each of 11 patients. We examined the effects of bile acid exposure on levels of MIRs 221 and 222 and CDX2 in EAC cells. We investigated the effects of inhibitors of MIRs 221 and 222 on growth of human EAC xenograft tumors in NOD/SCID/IL-2Rγnull mice.

Results

Levels of MIRs 221 and 222 increased and levels of p27Kip1 and CDX2 decreased in areas of EAC vs BE. Levels of MIRs 221 and 222 increased, along with activity of nuclear bile acid receptor/farnesoid X receptor (FXR), when cultured cells were exposed to bile acids. Incubation of cells with bile acids increased degradation of CDX2; this process was reduced when cells were also incubated with proteasome inhibitors. Overexpression of MIRs 221 and 222 reduced levels of p27Kip1 and CDX2, and knockdown of these MIRs increased levels of these proteins in cultured cells. Inhibitors of MIRs 221 and 222 increased levels of p27Kip1 and CDX2 in EAC cells and reduced growth of xenograft tumors in NOD/SCID/IL-2Rγnull mice.

Conclusions

We observed increased levels of MIRs 221 and 222 in human EAC tissues, compared with areas of BE from the same patient. We found that exposure of esophageal cells to bile acids activates FXR and increases levels of MIRs 221 and 222, reducing levels of p27Kip1 and promoting degradation of CDX2 by the proteasome. Our work opened the perspective of therapeutically targeting this pathway either via FXR antagonists or inhibitors of MIRs as a treatment option for BE and EAC.

Section snippets

Methods

Additional details are included in the Supplementary Material.

MIRs 221 and 222 Expression Was Higher in EAC Than in BE

We obtained 11 patients' paraffin-embedded tissues (10 men and 1 woman; mean age, 66.0 ± 8.8 years) of EAC. According to the Japanese classification of esophageal cancer (10th edition),20 the depth of tumor invasion was diagnosed as pT1b-SM2 in 1 patient, pT1b-SM3 in 3 patients, pT2-MP in 2 patients, and pT3-Ad in 5 patients. EAC area and surrounding BE (including dysplasia) area were dissected under microscopic observation (Supplementary Figure 1). Total RNA was extracted and quantitative

Discussion

Little is known about the molecular mechanism to down-regulate CDX2 in the carcinogenesis of BE. In addition, although the close relationship between BE and EAC has been well established, the pathogenic mechanism responsible for the transformation of BE to EAC is still far from properly understood, especially in view of the contribution of bile acids. Bile acids are known to cause oxidative stress and induce reactive oxygen species in esophageal squamous cells.28 In fact, cell proliferation was

Acknowledgments

The authors thank Dr Shinya Inoue for providing the FXR agonist and the TGR5 agonist, Dr Hiroshi Nakagawa for providing EPC1-hTERT cell line, Dr Rhonda Souza for providing BAR-T cell line, and Misa Kanekawa for providing general technical assistance. The preliminary results of this study were presented and awarded at the 4th meeting of Inflammation Conference in Alimentary Tract (ICAT) held in Tokyo on October 24, 2009, as well as at the United European Gastroenterology Week (UEGW) 2011 meeting.

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    Conflicts of interest The authors disclose no conflicts.

    Funding This study was supported by a Grant-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of Science (JSPS) (22300169, to HS), a grant from the Smoking Research Foundation (to HS), the Research Fund of Mitsukoshi Health and Welfare Foundation (to HS), the Grant from the JSPS Bilateral Joint Projects with Belgium (FWO; 11035231-000061, to HS), the Grant from the JSPS Bilateral Joint Projects with Korea (NRF; 12032211-000109, to H.S.), the Grant-in-Aid for JSPS Fellows (to JM), the Keio University Grant-in-Aid for Encouragement of Young Medical Scientists (to JM), the Graduate School Doctoral Student Aid Program, Keio University (to JM), the Ecole Polytechnique Fédérale de Lausanne (to JA), and the Swiss National Science Foundation (to JA).

    Author names in bold designate shared co-first authorship.

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